| This paper described the morphologic character and resource distribution of Stenoloma chusanum (Linn.)Ching, summarized the advances in pharmacological usage and chemical constituents about this plant, and discussed the biological activity of the major substance contained in this plant. The author suggested that the material basis of the biological activity of S. chusanum should be further studied and the techniques of introduction, propagation and cultivation should be investigated in order to establish the base for utilizing this plant. The main results of this research are as follows:1. Chemical constituents of Stenoloma chusanum ChingThe ethanol extracts from whole plant of S. chusanum were separated by column chromatography with silica gel, polyamide, Lichroprep RP-18and Sephadex LH20. Nineteen compounds were obtained and their structures were elucidated by means of chemical evidence, spectral analysis (ESI-MS,1H-NMR,13C-NMR, DEPT, HSQC, HMBC), and comparing with the data of literature and standard compounds. The compounds were stenolomaside A i.e.[3-(9,3’,5’-trimethox-4’-hydroxy-benzylidene) chroman-4-one]-5-O-β-D-glucoside, which exists E-(la) and Z-(lb) structures simultaneously; stenolin1i.e.4-O-β-D-(6-O-gentisoylglucopyranosyl) vanillic acid (2), stenolin2i.e.2-O-β-D-(6-O-gentisoylglucopyranosyl) gentisic acid (3), orientin (4), orientin-2"-O-β-D-glucopyranoside (5), vitexin (6), apigenin (7), vitexin rhamnoside (8), vitexin glucopyranoside (9), vanillic acid (10), syringic acid (11), vanillic acid (12), protocatechualdehyde(13),6,7-dihydroxycoumarin (14), pentadecanol (15), hexadecanoic acid (16),β-sitosterol (17), daucosterol (18). Among those compounds, Compounds(la),(1b),(2) and (3) were new compounds, compounds (4),(5),(8),(9),(10),(14),(15) and (16) were obtained from this plant for the first time.2. Analysis of main components in Stenoloma chusanum Ching and evaluation of the plant resourcesA method for determining orientin and vitexin from S. chusanum was established, content of the two flavonoids in different plants were measured and the plant resource were evaluated. Flavonoids were obtained by ultrasonic extraction, separation was carried out on Gemini C18column with a mixture of acetonitrile-1%phosphoric acid (V:V=15:85) as mobile phase and the flow rate was1.0mL/min, detective wavelength was340nm. Orientin showed a good linear relationship within0.025-0.148μg (R2=0.9995) and the average recovery was100.61%, RSD is2.18%. Vitexin showed a good linear relationship within0.035-0.207μg(R2=0.9999) and the average recovery was99.77%, RSD is2.37%. Stability experiment showed sample were stable within6h. Orientin and vitexin were the two main flavonoids and their ratio was variable in different plants according to the HPLC. The highest sample was from Qiubai Yunnan which contained orientin1.166mg/g and vitexin1.457mg/g, orientin decreased but vitexin increased when the plant was transplanted from Qiubai to Nanjing. Results showed that plant from Qiubai was an excellent resource based on the content of orientin and vitexin.Total flavonoids(TF) from the aerial part of Stenoloma chusanum in different province were extracted by ultrasonic extraction, with the standard curve established with rutin using NaNO2-Al(NO)3-NaOH as chromogenic agent, the TF were determined by spectrophotometer. Results showed that the highest plant sample was from Qiubai Yunnan, which contained TF as high as127.461mg·g-1and could keep the higher level though they were transplanted to Nanjing Jiangsu; TF in leaf blade was notably higher than in leaf stalk; sample cultivated with spores had high TF. It could be concluded that the plants from Qiubai was an excellent resource and the leaf blade should be collected mainly based on the TF, and it was practicable to utilize this plant resource by cultivating.3. Method of extraction and purification of orientin and vitexin from Stenoloma chusanumIn order to acquire pure orientin and vitexin from the aerial part of Stenoloma chusanum conveniently, safely and effectively, a method for extracting and separating the two flavonoids simultaneously was found. The essentials of this method were that:to get crude flavonoids precipitate at first, dissolve the precipitate with alkaline and dilute with water, neutralize the solution with acid, then separate the components by column chromatography with Lichroprep RP-18. The two flavonoids was obtained and their structures were elucidated by means of chemical evidence, spectral analysis (MS,1H-NMR,13C-NMR) and the data of literature. The purity of orientin and vitexin were detected with HPLC. Using the method described above; the target compounds of orientin and vitexin can be obtained with purity more than98%.4. Bioactivities of new compounds from Stenoloma chusanuma. Tests of antifungal activity about phenolic compoundsTwo new phenolic compounds, stenolin1, i.e.4-O-β-D-(6-O-gentisoylglucopyranosyl) vanillic acid, stenolin2, i.e.2-O-D-D-(6-O-gentisoylglucopyranosyl) gentisic acid, together with three known compounds, vanillic acid, syringic acid and gentisic acid were isolated from Stenoloma chusanum, the antifungal activities of these compounds were evaluated using test tube dilution method against Candida albicans, Cryptococcus neoformans, Trichophyton rubrum, Trichophyton mentagrophytes, Microsporum canis, Epidermophyton floccosum and Aspergillus niger. Results showed that the five compounds had the antifungal activity with MIC of25-100μg;·ml-1. Stenolin1and stenolin2, glucoside esters of phenolic compounds, had less antifungal activity than their simple phenolic compounds of vanillic acid and gentisic acid respectively.b. Inhibitory effects of flavonoids on the growth of human tumour cellsInhibitory effects of stenolomaside A, a new homoisoflavonoid extracted from Stenoloma chusanum, together with orientin and vitexin, two numerous compounds from the plant, on the growth of five kind of human tumour cells were tested at4levels of concentration of10-4ol/L,10-5mol/L,10-6mol/L and10-7mol/L with the method of MTT. Results showed that the three flavonoids all had weak or no effects on the tumour cells at high concentration. To human leukemic cell HL-60, phenolic acid had the inhibitory percentage of18.22%, orientin had less effect(13.29%) and vitexin had no effect. About human leukemic cell K562, orientin and vitexin had some inhibitory effects of19.29%and21.2%respectively, but stenolomaside A had petty effect of9.21%. Vitexin inhibited human lung adenocarcinoma cells A549with the percentage of18.99%, but stenolomaside A and orientin had no effects. All the three samples were almost ineffective on human hepatoma cells HepG2or SMMC-7721.5. Investigation of spore germination,gametophytes morphology and breeding technique with spore about Stenoloma chusanum ChingSpore germination of Stenoloma chusanum Ching were investigated. Effects of culture temperature, storage temperature, GA treatment and illumination intensity on spore germinating were studied using randomized blocks experiment and analysis of variance. Compared with room temperature (20-25℃), the germination velocity was higher at the culture temperature of28℃, although their maximal germination percentage was the same. Conserved temperature(A) significantly(P<0.01) affected spore germinating,-20℃reduced germination percentage. GA(B) did not affect germinating. Illumination intensity(C) significantly(P<0.01) affected germinating, there were no significant difference between full and weak illumination, but the dark reduced germination percentage. AxB, AxC, BxC and AxBxC had no significant interaction effect.The development of gametophytes of Stenoloma chusanum Ching was observed, and the method of breeding with spores were investigated. The morphology in the developmental stage from spore to sporophyllary leaves was observed with electron microscope, optical microscope and anatomical lens. Effects of temperature and illumination intensity on the development of gametophytes were surveyed in order to understand the proper breeding condition. Spores of S. chusanum are kidney-shaped with polar axis of (48-54)51μm and equatorial axis of (31-37)34μm, and their exine were smooth or had petty verrucous ornamentation.6days after planting, spores germinated and filament emerged then split to form the heart-shaped prothallium.45days after planting, antheridium developed at the ventral side and under the notch of prothallium.55days after planting, archegonium developed at the ventral side and under the notch of prothallium.12days after the formation of archegonium, a young sporophyllary appeared. With the culture substrate composed of peaty:garden soil:sand=2:1:1, under the temperature of about23℃, illumination intensity of10000Lx and saturated moisture, spores could reproduce sporophyllary. |
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